Improved PCR-based assays for pre-symptomatic diagnosis of light leaf spot and determination of mating type of Pyrenopeziza brassicae on winter oilseed rape

An existing PCR-based method for diagnosis of the winter oilseed rape (Brassica napus ssp oleifera) fungal pathogen Pyrenopeziza brassicae (cause of light leaf spot) was improved by the development of a pair of primers (PbN1 and PbN2) for use in nested-PCR reactions. The nested-PCR technique improved the detection of P. brassicae DNA in vitro by three orders of magnitude over that achieved using the first-round PCR primers (Pb1 and Pb2). In controlled environment experiments, the nested-PCR assay detected P. brassicae within infected B. napus leaves before visible light leaf spot symptoms developed and earlier than was possible by incubating infected leaves in polyethylene bags to promote sporulation of P. brassicae. A three-primer PCR technique using the primers PbM-1-3, PbM-2 and Mt3 was developed to distinguish between the two mating types (MAT-1 and MAT-2) of P. brassicae. This technique was able to determine the mating types present within DNA extracted from infected plant tissue, including tissue infected with both mating types together.